Triplet–triplet annihilation-based photon-upconversion to broaden the wavelength spectrum for photobiocatalysis

Photobiocatalysis is a growing field of biocatalysis. Especially light-driven enzyme catalysis has contributed significantly to expanding the scope of synthetic organic chemistry. However, photoenzymes usually utilise a rather narrow wavelength range of visible (sun)light. Triplet–triplet annihilation-based upconversion (TTA-UC) of long wavelength light to shorter wavelength light may broaden the wavelength range. To demonstrate the feasibility of light upconversion we prepared TTA-UC poly(styrene) (PS) nanoparticles doped with platinum(II) octaethylporphyrin (PtOEP) photosensitizer and 9,10-diphenylanthracene (DPA) annihilator (PtOEP:DPA@PS) for application in aqueous solutions. Photoexcitation of PtOEP:DPA@PS nanoparticles with 550 nm light led to upconverted emission of DPA 418 nm. The TTA-UC emission could photoactivate flavin-dependent photodecarboxylases with a high energy transfer efficiency. This allowed the photodecarboxylase from Chlorella variabilis NC64A to catalyse the decarboxylation of fatty acids into long chain secondary alcohols under green light (λ = 550 nm).

. CvFAP utilises a flavin cofactor, which in its photactivated form initiates the decarboxylation of carboxylic acids. As established previously, CvFAP can productively use wavelengths between 300 and 500 nm. Green light barely promotes CvFAP-catalysis. CvFAP and its mutants catalyse the irreversible decarboxylation of saturated and unsaturated fatty acids but also hydroxy fatty acids 15,18 , amino fatty acids, and ester bond-containing fatty acids (e.g., (Z)-11-(heptanoyloxy)undec-9-enoic acid) 19 , generating long chain secondary alcohols, long chain aliphatic amines and esters, respectively. Engineered variants of CvFAP have been reported for the conversion of short-chain carboxylic acids 22 as well as for the kinetic resolution of some α-substituted carboxylic acids and unnatural amino acid phosphinothricin 11,44 .
Overall, we envision UP to enlarge the wavelength scope of photo(bio)catalytic transformations such as the CvFAP-catalysed carboxylic acid decarboxylation (Fig. 1).

Results and discussion
Light upconversion by PS nanoparticles. TTA-UC nanoparticles were prepared by flash nanoprecipitation of 1.0 mL THF containing 1.0 wt% PS, 0.01 wt% PtOEP and 0.2 wt% DPA in stirred 9.0 mL milli-Q 45 . The PS nanoparticles were purified by repeated centrifugation and decantation of the supernatant. The prepared nanoparticles were spherical with an average diameter of 275 nm ( Figure S3). Efficiencies for encapsulation of PtOEP and DPA in PS were determined by UV-vis absorption spectroscopy to be 29% and 24%, respectively ( Figure S4 and Methods). These values correspond to molar concentrations of 3.5 μM (0.53 wt% relative to polymer) and 130 μM (8.9 wt% relative to polymer) for PtOEP and DPA, respectively. The TTA-UC nanoparticle suspension was stable for several days in air-equilibrated milli-Q water.
The PtOEP:DPA@PS nanoparticles exhibited blue emission with a peak wavelength of 418 nm, upon photoexcitation of PtOEP at a wavelength of 550 nm (Fig. 2a). The 418 nm emission was from DPA, because an identical emission spectrum was observed under direct photoexcitation of DPA at a wavelength of 394 nm. Note that 10 μM DPA (THF) did not produce fluorescence emission upon direct excitation at a wavelength of 550 nm, which rules out any unimolecular multi-photon fluorescence mechanism ( Figure S5). The photoluminescence excitation spectrum of the 418 nm emission possessed substantial contributions of PtOEP, corroborating the TTA-UC mechanism ( Figure S6). The corresponding anti-Stokes shift was 5807 cm −1 , typical of upconverted emission.
This UC emission involves two-photon processes, as seen from the quadratic dependence of its intensity on the photoexcitation power < 0.4 mW cm −2 (sky-blue region in Fig. 2b). The emission intensity became linearly proportional to the photoexcitation power > 0.4 mW cm −2 because the limiting step of TTA-UC changes from the bimolecular TTET or TTA processes to the unimolecular fluorescence transition (yellow region in Fig. 2b). The threshold photoexcitation power (i.e., 0.4 mW cm −2 ) is one order of magnitude smaller than those of the similar polymer UC nanoparticles of PtOEP and DPA 46,47 . The lower threshold photoexcitation power can be ascribed to the increased concentrations of the PtOEP and DPA dopants, specifically resulting in enhanced triplet-triplet energy transfer. The threshold photoexcitation power and the maximum upconversion efficiency remains invariant to repetitive photoexcitation ( Figure S7), which rules out photodegradation. The Φ UC , which was determined using a rhodamine B standard 48 , was found to increase in proportion with the photoexcitation power, and reached a saturated value of 2.1% ( Figure S9 and Methods). The Φ UC remained 2.0% under the photocatalysis reaction condition described below (i.e., a distance of 10 cm from the photon source (Xe lamp) and a photoexcitation power of 2.8 mW cm −2 ). The threshold photoexcitation power and Φ UC remained the same ) to be 57%. This Φ ET value is two-fold greater than that with free FAD (25%).
We also investigated energy transfer behaviors of free DPA (37 μM in THF) and DPA doped in PS nanoparticles (37 μM in PS) with FAD ( Figure S2). Our analyses with the standard Stern-Volmer equation revealed that doped DPA exhibited an Φ ET value (29%) greater than that (13%) of free DPA ( Figure S12). This improvement was likely ascribed to delocalisation of DPA exciton within the PS nanoparticles, and demonstrated the benefit of the nanoparticle approach. Collectively, our spectroscopic investigations revealed a high photoactivation ability of the TTA-UC nanoparticles.
Enzyme reactions under green light. The decarboxylation of ricinoleic acid (1) into (Z)-heptadec-9-en-7-ol (2) was used as a model reaction to examine application of TTA-UC for photoactivation of flavin-dependent photodecarboxylase (i.e., CvFAP) (Fig. 1). After CvFAP was added into the reaction medium containing 10 μM DPA in the form of ternary PtOEP:DPA@PS nanoparticles and 5 mM reaction substrates (1), green light www.nature.com/scientificreports/ (λ = 550 nm) was applied by a Xe lamp ( Figure S13). The decarboxylation products (2) were detected to 0.29 mM in the reaction medium at t = 420 min by GC/MS analysis (Table 1 and Figure S14a). This result indicated that the green light was upconverted into blue light (λ = 418 nm) by the PtOEP:DPA@PS nanoparticles, which led to photoexcitation of the FAD of CvFAP and subsequently decarboxylation of ricinoleic acid (1) into (Z)-heptadec-9-en-7-ol (2). The fatty alcohol (i.e., (Z)-heptadec-9-en-7-ol (2)) was also observed to 0.14 mM in the reaction medium without the nanoparticles ( Figure S14b), suggesting that FAD of CvFAP might be activated by green light irradiated by the Xe lamp. The turnover number (TON) of DPA in the nanoparticles and the enzyme was calculated to 15 and 25, respectively (Table 1), meaning that the light upconversion was 15 times achieved per molecule of DPA.
The TON of CvFAP (Table 1) was low in the light upconversion system as compared to the blue light-based reaction system 18 . One of the reasons may include formation of the reactive oxygen species (ROS) during light upconversion 41 , which may cause deactivation of the enzymes via oxidation of the sulfur-containing amino acids (e.g., cysteine 432, which is involved in catalysis) 49 .

Whole-cell reactions under green light.
Aiming at improving the enzyme reaction rates and TONs under green light, recombinant E. coli cells, which provide the ROS quenching systems (e.g., glutathione per-  (1), green light (λ = 550 nm) was irradiated by the Xe lamp (Figure S13). When 5 μM DPA was used for the light upconversion, (Z)-heptadec-9-en-7-ol (2) was produced to a rate of 2.5 μM/min, while the fatty alcohol (2) was produced to 1.4 μM/min without PS nanoparticles (Fig. 4a). This result indicated that (Z)-heptadec-9-en-7-ol (2) was produced to 1.1 μM/min via the light upconversion. Since the product concentrations reached 0.98 and 0.55 mM at t = 420 min in the presence and absence of the nanoparticles, the TONs of DPA and CvFAP at t = 420 min were estimated to 86 and 72, respectively, which are significantly greater than those of isolated enzyme reaction systems (Table 1). These results suggested that the FADs in the core of CvFAP enzymes, which were located in cytoplasm of the recombinant E. coli cells, had been quite efficiently excited by the PtOEP-DPA upconversion systems inside the poly(styrene) nanoparticles ( Figure S13).
The increase of DPA concentration in PtOEP:DPA@PS nanoparticles to 15 μM led to formation of (Z)heptadec-9-en-7-ol (2) to a rate of 3.2 μM/min (Fig. 4b). On the other hand, the fatty alcohol (2) was produced to 1.4 μM/min in the buffer without the PS nanoparticles, as in the experiment shown in Fig. 4b. In addition, the biotransformations in the buffer containing polystyrene nanoparticles only or DPA@PS nanoparticles only showed slightly lower product formation as compared to the biotransformation in the buffer without anything ( Figure S15). Thereby, it was assumed that the target product was produced in the reaction medium to at least 1.8 μM/min via the light upconversion. The reaction rate was approximately 60% greater than that of the reaction system including 5 μM DPA as annihilator. Increased DPA concentrations in PtOEP:DPA@PS nanoparticles did not alter the TTA-UC behaviors ( Figure S16). Thereby, it was assumed that the DPA concentrations have an influence on the photobiocatalytic reaction rates but did not linearly correlate.
The biotransformation of ricinoleic acid (1) into (Z)-heptadec-9-en-7-ol (2) was also carried out by the recombinant E. coli cells expressing CvFAP under blue light (λ = 450 nm). The reaction rate was significantly greater than under green light (λ = 550 nm) ( Figure S17), indicating that the PtOEP:DPA@PS nanoparticles need to be further improved. Thereby, the future study will focus on improvements of the TTA-UC system for enzymatic reactions in aqueous reaction systems.
Not only light-induced natural enzyme reactions but also light-dependent promiscuous enzymatic conversions have been extensively investigated for the environmentally benign synthesis of chemicals [1][2][3][4][5][8][9][10]15,[18][19][20] and fuels [21][22][23] . However, as flavin catalysts utilize only a part of the wavelength spectrum of visible light (e.g., λ = 450 nm), a significant part of the energy provided by visible light remains unused. Another issue of photobiocatalysis comprises the rather poor light penetration in traditional glass-batch reactors and external illumination 50,51 . Particularly blue light (λ = 450 nm) typically penetrates no more than a few millimeters to centimeters (especially in optically dense reaction mixtures). The problem should become more serious in scale-up of the photobiocatalysis. This study demonstrated that the PtOEP:DPA@PS-based TTU-AC system can be used to partially solve the poor penetration of blue light in photobiocatalytic reactors, by enabling the enzymes to use green light (λ = 550 nm) as a light source, which is capable of penetrating deeper into the core of bioreactors without damaging the enzyme biocatalysts.

Conclusions
This study demonstrated the effectiveness of the upconversion strategy toward steering photobiocatalysis. Upconverted fluorescence emission from TTA-UC poly(styrene) nanoparticles doped with the PtOEP photosensitiser and the DPA annihilator could photoactivated FAD and FAD-bound enzymes in aqueous solutions. Combination of TTA-UC nanoparticles and bacterial cells expressing CvFAP in aqueous reaction systems allowed to catalyse www.nature.com/scientificreports/ decarboxylation of fatty acids into secondary fatty alcohols under green light. The results will provide useful guidance to synthetic application of photobiocatalysis.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.